Transcription of NOD1 and NOD2 and their interaction with CARD9 and RIPK2 in IFN signaling in a perciform fish, the Chinese perch, Siniperca chuatsi.

NOD1 and NOD2 as two representative members of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family play important roles in antimicrobial immunity. However, transcription mechanism of nod1 and nod2 and their signal circle are less understood in teleost fish. In this study, with the cloning of card9 and ripk2 in Chinese perch, the interaction between NOD1, NOD2, and CARD9 and RIPK2 were revealed through coimmunoprecipitation and immunofluorescence assays. The overexpression of NOD1, NOD2, RIPK2 and CARD9 induced significantly the promoter activity of NF-κB, IFNh and IFNc. Furthermore, it was found that nod1 and nod2 were induced by poly(I:C), type I IFNs, RLR and even NOD1/NOD2 themselves through the ISRE site of their proximal promoters. It is thus indicated that nod1 and nod2 can be classified also as ISGs due to the presence of ISRE in their proximal promoter, and their expression can be mechanistically controlled through PRR pathway as well as through IFN signaling in antiviral immune response.

Transcriptome analyses in juvenile yellow perch (Perca flavescens) exposed in vivo to clothianidin and chlorantraniliprole: Possible sampling bias.

The St. Lawrence River is an important North American waterway that is subject to anthropogenic pressures including intensive urbanization, and agricultural development. Pesticides are widely used for agricultural activities in fields surrounding the yellow perch (Perca flavescens) habitat in Lake St. Pierre (Quebec, Canada), a fluvial lake of the river where the perch population has collapsed. Clothianidin and chlorantraniliprole were two of the most detected insecticides in surface waters near perch spawning areas. The objectives of the present study were to evaluate the transcriptional and biochemical effects of these two pesticides on juvenile yellow perch exposed for 28d to environmental doses of each compound alone and in a mixture under laboratory/aquaria conditions. Hepatic mRNA-sequencing revealed an effect of chlorantraniliprole alone (37 genes) and combined with clothianidin (251 genes), but no effects of clothianidin alone were observed in perch. Dysregulated genes were mostly related to circadian rhythms and to Ca2+ signaling, the latter effect has been previously associated with chlorantraniliprole mode of action in insects. Moreover, chronic exposure to clothianidin increased the activity of acetylcholinesterase in the brain of exposed fish, suggesting a potential non-target effect of this insecticide. Further analyses of three clock genes by qRT-PCR suggested that part of the observed effects of chlorantraniliprole on the circadian gene regulation of juvenile perch could be the result of time-of-day of sacrifice. These results provide insight into biological effects of insecticides in juvenile perch and highlight the importance of considering the circadian rhythm in experimental design and results analyses.

Heterologous Exchanges of Glycoprotein and Non-Virion Protein in Novirhabdoviruses: Assessment of Virulence in Yellow Perch (Perca flavescens) and Rainbow Trout (Oncorhynchus mykiss).

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses in two different species belonging to the Novirhabdovirus genus. IHNV has a narrow host range restricted to trout and salmon species, and viruses in the M genogroup of IHNV have high virulence in rainbow trout (Oncorhynchus mykiss). In contrast, the VHSV genotype IVb that invaded the Great Lakes in the United States has a broad host range, with high virulence in yellow perch (Perca flavescens), but not in rainbow trout. By using reverse-genetic systems of IHNV-M and VHSV-IVb strains, we generated six IHNV:VHSV chimeric viruses in which the glycoprotein (G), non-virion-protein (NV), or both G and NV genes of IHNV-M were replaced with the analogous genes from VHSV-IVb, and vice versa. These chimeric viruses were used to challenge groups of rainbow trout and yellow perch. The parental recombinants rIHNV-M and rVHSV-IVb were highly virulent in rainbow trout and yellow perch, respectively. Parental rIHNV-M was avirulent in yellow perch, and chimeric rIHNV carrying G, NV, or G and NV genes from VHSV-IVb remained low in virulence in yellow perch. Similarly, the parental rVHSV-IVb exhibited low virulence in rainbow trout, and chimeric rVHSV with substituted G, NV, or G and NV genes from IHNV-M remained avirulent in rainbow trout. Thus, the G and NV genes of either virus were not sufficient to confer high host-specific virulence when exchanged into a heterologous species genome. Some exchanges of G and/or NV genes caused a loss of host-specific virulence, providing insights into possible roles in viral virulence or fitness, and interactions between viral proteins.

Informing the Exposure Landscape: The Fate of Microplastics in a Large Pelagic In-Lake Mesocosm Experiment.

Understanding microplastic exposure and effects is critical to understanding risk. Here, we used large, in-lake closed-bottom mesocosms to investigate exposure and effects on pelagic freshwater ecosystems. This article provides details about the experimental design and results on the transport of microplastics and exposure to pelagic organisms. Our experiment included three polymers of microplastics (PE, PS, and PET) ranging in density and size. Nominal concentrations ranged from 0 to 29,240 microplastics per liter on a log scale. Mesocosms enclosed natural microbial, phytoplankton, and zooplankton communities and yellow perch (Perca flavescens). We quantified and characterized microplastics in the water column and in components of the food web (biofilm on the walls, zooplankton, and fish). The microplastics in the water stratified vertically according to size and density. After 10 weeks, about 1% of the microplastics added were in the water column, 0.4% attached to biofilm on the walls, 0.01% within zooplankton, and 0.0001% in fish. Visual observations suggest the remaining >98% were in a surface slick and on the bottom. Our study suggests organisms that feed at the surface and in the benthos are likely most at risk, and demonstrates the value of measuring exposure and transport to inform experimental designs and achieve target concentrations in different matrices within toxicity tests.

Differing physiological performance of coexisting cool- and warmwater fish species under heatwaves in the Midwestern United States.

Heatwaves are becoming more frequent and intensified with climate change. Freshwater ecosystems are among the most threatened, within which, differing responses between cool- and warmwater species to heatwaves can lead to fundamental changes in communities. Physiological experiments can identify potential mechanisms underlying the impacts of such heatwaves on fish communities. In the current study, we quantified the oxygen consumption rate, aerobic scope and swimming performance of cool- and warmwater fish species following the simulation of short-term heatwaves currently occurring in streams in the Midwestern United States. The coolwater predator walleye (Sander vitreus) showed clear thermal disadvantages relative to the warmwater predator largemouth bass (Micropterus salmoides), based on a high metabolic cost during the heatwave, low metabolic activity when encountering prey, and reduced swimming performance following the heatwave. Largemouth bass also showed a thermal advantage relative to the warmwater prey fathead minnow (Pimephales promelas) related to swimming performance and energetic costs, highlighting differing thermal responses between predators and prey. This study demonstrates the importance of considering short-term extreme thermal events in the response of aquatic communities to climate stressors.

Paternal-effect-genes revealed through sperm cryopreservation in Perca fluviatilis.

Knowledge about paternal-effect-genes (PEGs) (genes whose expression in the progeny is influenced by paternal factors present in the sperm) in fish is very limited. To explore this issue, we used milt cryopreservation as a specific challenge test for sperm cells, thus enabling selection amidst cryo-sensitivity. We created two groups of Eurasian perch (Perca fluviatilis) as a model - eggs fertilized either with fresh (Fresh group) or cryopreserved (Cryo group) milt from the same male followed by phenotypic-transcriptomic examination of consequences of cryopreservation in obtained progeny (at larval stages). Most of the phenotypical observations were similar in both groups, except the final weight which was higher in the Cryo group. Milt cryopreservation appeared to act as a "positive selection" factor, upregulating most PEGs in the Cryo group. Transcriptomic profile of freshly hatched larvae sourced genes involved in the development of visual perception and we identified them as PEGs. Consequently, larvae from the Cryo group exhibited enhanced eyesight, potentially contributing to more efficient foraging and weight gain compared to the Fresh group. This study unveils, for the first time, the significant influence of the paternal genome on the development of the visual system in fish, highlighting pde6g, opn1lw1, and rbp4l as novel PEGs.

Taxonomic revision of the Pirate Perches, Aphredoderus, (Percopsiformes: Aphredoderidae) with descriptions of two new species.

Pirate Perches, Aphredoderus, are a widespread lowland freshwater fish native to the Eastern half of the United States. Aphredoderus was thought to contain a single species divided into an Eastern and Western subspecies on either side of the Appalachian Mountains with a widespread intergrade zone through much of the Eastern Gulf of Mexico and Southern Atlantic drainages. We use morphology and genetic data from specimens spanning the entire range of the genus to determine species limits within Aphredoderus. We find evidence of five species, four of which exhibit widespread sympatry in the Southeastern United States. We elevate A. sayanus sayanus and A. sayanus gibbosus to species, redescribe A. mesotrema, supplement previous descriptions, and describe two new species, A. retrodorsalis, and A. ornatus.

Water quality in a shallow eutrophic lake is unaffected by extensive thinning of planktivorous and benthivorous fish species.

Ambitious to fulfill the European Water Framework Directive obligations, the European governments support projects to rehabilitate lakes with poor water quality. However, most lake restorations having relied on biomanipulation by fish thinning have failed to improve or even maintain water quality. Previous attempts removed all target fish species simultaneously, thus making it impossible to assess the specific impact of each feeding group on water chemistry. Lake Bromme was selected for extensive, time-selective fish biomanipulation to improve water clarity and promote submerged macrophytes and piscivorous fish stocks over a three-year monitoring period. Thinning of adult benthivorous bream (Abramis brama) and tench (Tinca tinca) was conducted throughout year one while thinning in years two and three targeted planktivorous roach (Rutilus rutilus), juvenile bream, and small perch (Perca fluviatilis). Yearly fish surveys assessed changes in fish population structure and biomass. Water quality parameters were monitored continually, and the cover of submerged macrophytes was surveyed annually via sonar. We found no improvement in water clarity or reductions of nutrients, organic particles, chlorophyll concentrations, or watercolor, despite a 6-fold thinning of total estimated fish biomass, from 112 to 19 kg ha-1. Over the period, the macrophyte cover increased from 0.8 to 13.5 %, but no recruitment of large piscivorous fish (perch and pike (Esox lucius) > 10 cm) was detected. We found higher correlations of particle concentration and water clarity to water temperature than to wind speed, which indicates sediment particle resuspension by the remaining fish community (mostly carp Cyprinus carpio) that forage on benthos in shallow lakes. Further system-ecological research in Lake Bromme should evaluate whether thinning the stock of carp and increasing plant cover may improve water quality and test which optical properties sustain high water turbidity and prevent shallow, eutrophic lakes like Lake Bromme from responding to intense fish thinning.

Integrating fish swimming abilities into rapid road crossing barrier assessment: Case studies in the southeastern United States.

Many aquatic networks are fragmented by road crossing structures; remediating these barriers to allow fish passage is critical to restoring connectivity. Maximizing connectivity requires effective barrier identification and prioritization, but many barrier prioritization efforts do not consider swimming capabilities of target species. Given the many potential barriers within watersheds, inventory efforts integrating species-specific swimming speeds into rapid assessment protocols may allow for more accurate barrier removal prioritization. In this study, we demonstrate an approach for integrating fish swimming speeds into rapid barrier assessment and illustrate its utility via two case studies. We measured critical swimming speeds (Ucrit) of two stream-resident fish species with very different swimming modes: Yoknapatawpha Darter (Etheostoma faulkneri), an at-risk species whose current distribution is restricted to highly degraded habitat, and Bluehead Chub (Nocomis leptocephalus), an important host species for the federally endangered Carolina Heelsplitter mussel (Lasmigona decorata). We assessed potential barriers for Yoknapatawpha Darters in the Mississippi-Yocona River watershed, and Bluehead Chubs in the Stevens Creek watershed, South Carolina, USA. We integrated Ucrit into the Southeast Aquatic Resources Partnership (SARP) barrier assessment protocol by estimating the proportion of individuals per species swimming at least as fast as the current through the assessed structures. Integrating Ucrit estimates into the SARP protocol considerably increased barrier severity estimates and rankings only for Yoknapatawpha Darters in the Yocona River watershed. These results indicate the importance of including species-specific swimming abilities in rapid barrier assessments and the importance of species-watershed contexts in estimating where swimming speed information might be most important. Our method has broad application for those working to identify barriers more realistically to improve species-specific fish passage. This work represents a next step in improving rapid barrier assessments and could be improved by investigating how results change with different measurements of swimming abilities and structure characteristics.

Sea perch (Lateolabrax japonicus) UBC9 augments RGNNV infection by hindering RLRs-interferon response.

Small ubiquitin-like modifier (SUMO) is a reversible post-translational modification that regulates various biological processes in eukaryotes. Ubiquitin-conjugating enzyme 9 (UBC9) is the sole E2-conjugating enzyme responsible for SUMOylation and plays an important role in essential cellular functions. Here, we cloned the UBC9 gene from sea perch (Lateolabrax japonicus) (LjUBC9) and investigated its role in regulating the IFN response during red-spotted grouper nervous necrosis virus (RGNNV) infection. The LjUBC9 gene consisted of 477 base pairs and encoded a polypeptide of 158 amino acids with an active site cysteine residue and a UBCc domain. Phylogenetic analysis showed that LjUBC9 shared the closest evolutionary relationship with UBC9 from Paralichthys olivaceus. Tissue expression profile analysis demonstrated that LjUBC9 was significantly increased in multiple tissues of sea perch following RGNNV infection. Further experiments showed that overexpression of LjUBC9 significantly increased the mRNA and protein levels of RGNNV capsid protein in LJB cells infected with RGNNV, nevertheless knockdown of LjUBC9 had the opposite effect, suggesting that LjUBC9 exerted a pro-viral effect during RGNNV infection. More importantly, we found that the 93rd cysteine is crucial for its pro-viral effect. Additionally, dual luciferase assays revealed that LjUBC9 prominently attenuated the promoter activities of sea perch type Ⅰ interferon (IFN) in RGNNV-infected cells, and overexpression of LjUBC9 markedly suppressed the transcription of key genes associated with RLRs-IFN pathway. In summary, these findings elucidate that LjUBC9 impairs the RLRs-IFN response, resulting in enhanced RGNNV infection.

Effect of type and concentration of antioxidant on sperm motility, viability, and DNA integrity of climbing perch Anabas testudineus Bloch, 1792 (Pisces: Anabantidae) post-cryopreservation.

Sperm quality is preserved through the crucial involvement of antioxidants, which play a vital role in minimizing the occurrence of reactive oxygen species (ROS) during the cryopreservation process. The suitability of the type and concentration of antioxidants are species-dependent, and this study is crucial in order to improve the quality of the climbing perch sperm post-cryopreservation. Therefore, this study aimed to determine the best type and concentration of antioxidants for cryopreservation of climbing perch Anabas testudineus sperm. To achieve this, 6 types of antioxidants, namely, ascorbic acid, beta-carotene, glutathione, butylated hydroxytoluene (BHT), myo-inositol, and alpha-tocopherol, with inclusion of a control were tested in 3 replications at three concentration levels of 0 mg/L (control), 20 mg/L, 40 mg/L, and 60 mg/L. Sperm was diluted in a glucose-base extender at a ratio of 1:60 (sperm: glucose base), then 10 % DMSO and 5 % egg yolk was added before cryopreservation for two weeks. The results showed that the type and concentration of antioxidants had a significant effect on the motility and viability of cryopreserved climbing perch sperm (P < 0.05), where the best results for ascorbic acid, beta-carotene, glutathione, myo-inositol, and alpha-tocopherol were obtained at a concentration of 60 mg/L, while BHT was at a concentration of 20 mg/L. The best results for glutathione, myo-inositol, and alpha-tocopherol were significantly different from other treatments, while the best results for ascorbic acid and beta-carotene (60 mg/L) were not significantly different from the 40 mg/L concentration, while the best results for BHT were not significantly different from the control treatments. Therefore, the best concentration of glutathione, myo-inositol, and alpha-tocopherol was 60 mg/L, while for ascorbic acid and beta-carotene it was 40 mg/L, and BHT was not recommended. DNA integrity analysis indicated the absence of fragmentation in all samples, including fresh, control, and treated sperm. Based on practical and economic considerations, myo-inositol at 60 mg/L was recommended for cryopreservation of climbing perch A. testudineus sperm.

Haematological, biochemical, enzymological changes and mitochondrial dysfunction of liver in freshwater climbing perch Anabas testudineus during their acute and chronic exposure to sodium fluoride.

Anthropogenic activities are increasing fluoride concentration in watercourses. The present study focuses on the sublethal toxicity of sodium fluoride during sub-chronic and chronic time periods in the freshwater fish Anabas testudineus. The 96-hour LC50 value for fluoride was found to be 616.50 mg/L. Excessive mucous production and hyper excitability, followed by loss of balance, were seen in fish under acute fluoride exposure. Significant reduction in yield and specific growth rate of fish were assessed at 15, 30 and 45-days exposure intervals. Different bio-indicators like Hepatosomatic-index, Gonadosomatic-index and fecundity were reduced significantly in fish exposed to 10% (61.6 mg/L) and 20% (123.2 mg/L) of 96 h of LC50 values of fluoride in comparison to control. Toxicant concentrations directly correlated with parameter lowering. Fluoride exposure increased plasma glucose, creatinine, AST, and ALT and reduced total RBC, haemoglobin content, Hct (%), plasma protein, and cholesterol. Moreover, fluoride exposure significantly reduces the mitochondrial membrane potential in liver. This may result in metabolic depression, haematological, biochemical, and enzymological stress. The in-silico structural analysis predicts that fluoride may impede cytochrome c oxidase of the electron transport system, hence inhibiting mitochondrial functionality. These findings collectively highlight the urgent need for stringent regulation and monitoring of fluoride levels in freshwater ecosystems, as the subchronic and chronic effects observed in A. testudineus may have broader implications for aquatic ecosystems.

Temporal Trends of Great Lakes Legacy Contaminants: Ecological and Biological Considerations Applying the Age-Trend Model.

The USEPA Great Lakes Fish Monitoring and Surveillance Program (GLFMSP) has been monitoring top predator lake trout and walleye contaminant concentrations since the early 1970s. Our research revealed that select legacy contaminant groups (∑PCBs, ∑DDTs, ∑chlordanes, and ∑5PBDEs) have similar t1/2 and k2 values across the Great Lakes, with the exception of both Lake Erie sites and the Lake Superior─Keweenaw Point site. The slower halving times determined at both Lake Erie sites are consistent with legacy contaminant remobilization due to extreme weather climate effects and past remedial actions on the Detroit River, whereas the Lake Superior─Keweenaw Point site demonstrates contaminant halving times approaching the exponential minimum. Overall, Great Lakes select contaminant groupings have decreased between 25.8 and 97.9% since 2004. An age-normalized Great Lakes Contaminant Index (GLCI) was devised, indicating both Lake Michigan sites as the most highly impacted. The mean absolute deviation statistic was applied, documenting the need to age-correct contaminant trends due to highly variable age profiles. With the noted exceptions, the uniformity of age-corrected trend modeling suggests that a combination of the fundamental biological and physicochemical mechanisms of natural contaminant sequestration, declining dissolved water concentrations, accumulation/metabolism/depuration, and the overall reduction of legacy contaminant loading are driving the generally consistent rates of declines in the Great Lakes. Many of the biological and ecological stressors currently associated with climate change appear to be accounted for by the age-trend model.

Increasing mercury bioaccumulation and biomagnification rates of Nile perch (Lates niloticus L.) in Winam Gulf, Lake Victoria, Kenya.

The Nile perch (Lates niloticus L.) commercial fishery for Lake Victoria in East Africa is an important source of revenue and employment. We focused on shifts in food web structure and total mercury (THg) bioaccumulation and biomagnification in Nile perch, and lower food web items collected from Winam Gulf (Kenya) sampled 24 years apart (1998 and 2022). Stable isotope carbon (δ13C) values were higher in all species from 2022 compared to 1998. Stable nitrogen isotope (δ15N) values in baseline organisms were lower in 2022 compared to 1998. In Nile perch, δ15N values were correlated with total length, but the δ15N-length regressions were steeper in 1998 compared to 2022 except for one large (158 cm) Nile perch from 1998 with an uncharacteristically low δ15N value. Total Hg concentrations were lower in lower trophic species from 2022 compared to 1998. However, the THg bioaccumulation rate (as a function of fish length) in Nile perch was greater in 2022 compared to 1998 resulting in 24.2 % to 42.4 % higher wet weight dorsal THg concentrations in 2022 Nile perch for market slot size (50 to 85 cm) fish. The contrasting observations of increased THg bioaccumulation with size in 2022 against decreases in the rate of trophic increase with size and lower THg concentrations of lower food web items imply reduced fish growth and potential bioenergetic stressors on Winam Gulf Nile perch. All samples except 1 large Nile perch (139 cm total length collected in 2022) had THg concentrations below the European Union trade limit (500 ng/g wet weight). However, for more vulnerable individuals (women, children and frequent fish eaters), we recommend a decrease in maximum monthly meal consumption for 55-75 cm Nile perch from 16 meals per month calculated for 1998 to a limit of 8 meals per month calculated for 2022.

Dietary supplementation of VA enhances growth, feed utilization, glucose and lipid metabolism, appetite, and antioxidant capacity of Chinese perch (Siniperca chuatsi).

The aim of this study was to investigate the effect of dietary vitamin A on juvenile Chinese perch (Siniperca chuatsi). Chinese perch were fed with five experimental diets containing 0, 20, 40, 60, and 80 mg VA·kg-1 for 8 weeks. Results showed that dietary vitamin A significantly influenced the fish's growth, feed utilization, glucose and lipid metabolism, appetite, and antioxidant capacity. Vitamin A-supplemented groups had higher weight gain rate (WGR) and specific growth rate (SGR) compared to the control group. Feed conversion ratio (FCR) was also lower in the vitamin A-supplemented groups. Dietary vitamin A had no significant effect on the survival rate (SR). Compared to the control group, fish fed with vitamin A had increased feed intake (FI), and the expression of appetite-promoting genes (npy and agrp) was significantly higher in the 40 mg VA·kg-1 group. Vitamin A also enhanced the utilization of dietary protein by Chinese perch. The serum glucose content of the fish fed with 40 mg VA·kg-1 diet was significantly higher than that of the control group and 20 mg VA·kg-1 diet, indicating that the promoting effect of VA on gluconeogenesis was greater than that on glycolysis. Additionally, dietary vitamin A increased the expression of lipid metabolism-related genes (hl and fas) and antioxidant genes (nrf2 and gpx) in the fish. These results suggest that the optimal vitamin A requirement of juvenile Chinese perch bream was estimated to be 37.32 mg VA·kg-1 based on broken-line regression analysis of WGR. In conclusion, this study provides valuable insights into the potential benefits of dietary vitamin A on the growth, metabolism, and antioxidant capacity of Chinese perch.

DNA metabarcoding reveals spatial and temporal variation of fish eye fluke communities in lake ecosystems.

Eye flukes (Diplostomidae) are diverse and abundant trematode parasites that form multi-species communities in fish with negative effects on host fitness and survival. However, the environmental factors and host-related characteristics that determine species diversity, composition, and coexistence in such communities remain poorly understood. Here, we developed a cost-effective cox1 region-specific DNA metabarcoding approach to characterize parasitic diplostomid communities in two common fish species (Eurasian perch and common roach) collected from seven temperate lakes in Estonia. We found considerable inter- and intra-lake, as well as inter-host species, variation in diplostomid communities. Sympatric host species characterization revealed that parasite communities were typically more diverse in roach than perch. Additionally, we detected five positive and two negative diplostomid species associations in roach, whereas only a single negative association was observed in perch. These results indicate that diplostomid communities in temperate lakes are complex and dynamic systems exhibiting both spatial and temporal heterogeneity. They are influenced by various environmental factors and by host-parasite and inter-parasite interactions. We expect that the described methodology facilitates ecological and biodiversity research of diplostomid parasites. It is also adaptable to other parasite groups where it could serve to improve current understanding of diversity, distribution, and interspecies interactions of other understudied taxa.

Male recognition of conspecific female chemical cues in a diverse clade of freshwater fishes.

Where orangethroat darters (Etheostoma: Ceasia) and rainbow darters (Etheostoma caeruleum) co-occur, males prefer conspecific over heterospecific females. The cues males use to identify conspecific females remain unclear. We conducted behavioral trials to ask whether chemical cues function in conspecific recognition. We found that males from three orangethroat darter species preferentially associate with female scent over a control. Our results support the use of olfaction in conspecific identification in the orangethroat clade and contribute to our understanding of signals that may facilitate species recognition and underlie the evolution of behavioral isolation.

Riverscape genetics of the orangethroat darter complex.

Freshwater darters belonging to the orangethroat darter species complex, or Ceasia, are widely distributed in the Central and Southern United States, with ranges that span both glaciated and unglaciated regions. Up to 15 species have been recognized in the complex, with one, Etheostoma spectabile, having a widespread northern distribution and another, Etheostoma pulchellum, having a sizeable southern distribution. The other species in the complex have much more restricted distributions in unglaciated regions of the Central Highlands. We sampled 384 darters from 52 sites covering much of the range of Ceasia and evaluated patterns of genetic diversity, genetic structure, and pre- and post-glacial patterns of range contraction and expansion. We anticipated finding much stronger signals of genetic differentiation and diversification in unglaciated regions, given the higher species diversity and levels of endemism reported there. Surprisingly, microsatellite genotyping revealed two well-differentiated genetic clusters of E. spectabile in samples from glaciated regions, one confined to the Illinois River basin and another found in the Wabash drainage and Great Lakes tributaries. This suggests that there was expansion from two isolated glacial refugia, with little subsequent post-glacial gene flow. Fish collected from throughout the unglaciated region were less genetically differentiated. Fish assigned to Etheostoma burri and Etheostoma uniporum based on collection sites and morphological characters were not genetically differentiated from E. spectabile samples from the region. Hybridization and introgression occurring in the Central Highlands may confound genetic delineation of species in this region of high endemism and diversity.

Measuring Total Mercury Through Freeze-Thaw Cycles.

Total mercury (THg) analysis of fish tissue requires the samples to be frozen for long-term storage or analyzed immediately after the fish sample has been obtained. Such rigid field sampling protocols do not account for the logistical challenges that can occur if sampling is conducted in remote areas or collaboratively via community-based monitoring initiatives. Here we present a study that examines the loss of THg from Walleye tissue that has thawed for 3 and 5 days, mimicking potential thaw during transport from remote locations. THg concentrations were not significantly influenced (Kruskal-Wallace, p-value > 0.05) by the thaw and refreeze events. The mean THg concentration per day group was 0.67 ug/g, SD = 0.03, 0.65 ug/g, SD = 0.01, and 0.72 ug/g, SD = 0.01 for the control (no thaw), 3-day thaw and 5-day thaw, respectively. Results from this study suggest the analytical integrity of samples may be maintained if freeze-thaw situations arise when conducting community-based mercury monitoring programs.

Description, life cycle, and phylogenetics of Proterometra wigglewomble n. sp. (Digenea: Azygiidae) from the Cahaba River, Alabama, U.S.A.

We herein describe Proterometra wigglewomble n. sp. (Digenea: Azygiidae: Azygiinae) from the Cahaba River, Alabama, USA, which asexually reproduces in the compact elimia, Elimia showalteri (Lea, 1860) (Cerithioidea: Pleuroceridae) and matures in the oesophagus of the blackbanded darter, Percina nigrofasciata (Agassiz, 1854) (Perciformes: Percidae). Adults of the new species differ from congeners by having a small body and eggs having a wholly fimbriated surface that appears as a cilia-like brush border. Live naturally-shed cercariae of the new species differ from those of its congeners by having a strongly claviform tail stem bearing aspinose mammillae, a single furca, excretory pores that open on the posterior margin of the single furca, and few eggs in the cercarial distome. The behaviour of the cercaria further differentiates the new species. Naturally-shed cercariae of P. wigglewomble secrete a jelly-like adhesive that coats the surface of the furca and evidently facilitates attachment to the surface of glass, plastic, and snail shell. Attached cercariae vigorously wiggle and thrash about once attached, as if mimicking the larva of a stream insect so as to lure the blackbanded darter to eat it. Phylogenetic analyses recovered monophyletic Azygiidae, comprising monophyletic Leuceruthrinae Goldberger, 1911 and polyphyletic Azygiinae Lühe, 1909. The present study is the largest taxon sampling for Azygiidae and the first to include 28S sequences of Leuceruthrus. Compact elimia and blackbanded darter are new host records for Proterometra. The new species is the 3rd congener reported from the Cahaba River, a region renowned for its fish and snail endemic biodiversity.